The goal of this proposal is to study mechanisms of transport across the nuclear membrane using the HIV-1 proteins Vpr and Rev as model import and export systems. Part I of this proposal will focus on characterization of HIV-1 Vpr nuclear import. Vpr is thought to possess a novel nuclear targeting signal and consequently may use a transport pathway different from that of proteins containing a classical nuclear localization signal (NLS), as exemplified by the SV40 large T antigen sequence. A variety of cell-based assays will be used to delineate the import pathway used by Vpr into the cell nucleus. Part II of this proposal is an investigation of the function of hRip, a putative transport factor implicated in the export of HIV-1 Rev from the nucleus. hRip is a Rev-interacting protein proposed to be a component of the cellular export machinery. A tissue culture cell line deficient in the RIP gene will be constructed and analyzed functionally and biochemically to delineate the role of Rip in Rev-mediated export of HIV mRNA and export of other RNA species. New insight will be gained from these studies about the various import and export processes used by proteins and RNA in regulated nucleocytoplasmic trafficking. Because the ability to actively traverse the nuclear membrane in both directions is essential to viral replication, mechanisms used by HIV for proper cellular localization during its life cycle may be identified as potential therapeutic targets.